Yijie Geng

Effect of polymorphisms of endothelial nitric oxide synthase on ischemic stroke: a case-control study in a Chinese population.

BACKGROUND Nitric oxide (NO) synthesized by endothelial nitric oxide synthase (eNOS) plays a key role in vascular regulation and atherosclerosis, therefore, eNOS may be a candidate gene for ischemic stroke (IS). However, it is still controversial whether eNOS polymorphisms are a risk factor for IS. METHODS Three polymorphisms of the eNOS gene (-922A/G, -786T/C, 894G/T) were determined by using TaqMan SNP genotyping assay in 309 Chinese patients with IS and 309 Chinese controls. RESULTS The frequency of eNOS -922 G allele was significantly higher in the patients than the controls (12.14% vs 8.09%, P=0.018). The distribution of eNOS genotypes differed insignificantly between the 2 groups. The frequency of the eNOS -786 CC genotype was higher in the patients than the controls (OR=3.819, P=0.029). With respect to -922A/G, the AG+GG genotype increased the risk for IS (OR=1.523, P=0.047). After adjustment for confounding factors, the odds ratios of -786 CC and -922 variant genotype (AG+GG) for IS were 4.580 and 1.656, respectively. However, haplotype analysis revealed the frequencies of Hap4 (GCG) and Hap7 (GCT) were significantly higher in the patients than the controls (P=0.035, 0.042). CONCLUSIONS eNOS -922A/G and -786T/C may affect the susceptibility to IS and certain haplotypes of the eNOS gene may be associated with a higher susceptibility to IS.

Epidemiological study on Clonorchis sinensis infection in Shenzhen area of Zhujiang delta in China.

To study the transmission route and epidemiological features of Clonorchis sinensis infection in Shenzhen area, which is the biggest immigration city in the south of China, we examined 1,473 individuals (710 males and 763 females) to assess the current status of C. sinensis infection among the people in a village of Shenzhen in Zhujiang delta of Guangdong province, China. Freshwater snails, 630, of different species known as the first intermediate host of C. sinensis were collected and examined for cercaria infection, and 430 freshwater fishes of different species as the second intermediate host were examined for metacercaria infection. Among 1,473 people examined, 70 (4.75%) were found infected with C. sinensis. By counting eggs per gram feces (EPG), it was found that the intensity of infection in males was stronger than that of females, and the average EPG was 41.87 in all population. Snails, 1.15%, were infected with cercariae of C. sinensis. The average infection rate of freshwater fishes of 15 species with metacercariae of C. sinensis was 16.97%, and the carps reached the highest infection rate (40.74%). A questionnaire was designed with 12 questions covering socioeconomic conditions and human behavior, contamination of the environment, and fishponds. Of 1,473 interviewees, 54% did not know about fluke disease or its transmission route, 12% of those who knew about the fluke believed that the infection causes no harm or only slight harm to their health. Of the interviewees, 27%, ate raw fish at least one to two times per month. Of families, 5% used the same utensils for both raw fish and cooked food. Of the fishpond owners, 40% fed their fishes with feces of domestic animals and humans. All these factors of unhealthy behaviors, poor knowledge, inappropriate farming/fishery practices, and eating raw fish have made the prevalence of clonorchiasis increase in humans in the Shenzhen area. It is urgent to perform a control program, including health education, environmental modification, reform of traditional farming/fishery practice, mass screening, and chemotherapy for humans, and the management of domestic animals to decrease C. sinensis infection in the human population in Shenzhen.

Association of the Genetic Polymorphisms in Pre-MicroRNAs with Risk of Ischemic Stroke in a Chinese Population

microRNA (miRNA) plays a role in the pathogenesis of ischemic stroke, and single nucleotide polymorphisms in miRNA genes may contribute to disease susceptibility. However, the effect of miR-146a, miR-196a2, and miR-499 polymorphisms on ischemic stroke susceptibility has been rarely reported. Using the TaqMan assay, we evaluated the association of hsa-miR-146a/rs2910164, hsa-miR-196a2/rs11614913, and hsa-miR-499/rs3746444 polymorphisms with the risk of ischemic stroke in a Chinese population with 531 ischemic stroke patients and 531 control subjects. Rs2910164 C/G genotypes were significantly associated with increased risk of ischemic stroke in different genetic model (homozygote comparison: OR = 2.00, 95% CI, 1.29–3.12, P = 0.002; additive model: OR = 1.35, 95% CI, 1.10–1.65, P = 0.004;dominant model: OR = 1.33, 95% CI, 1.00–1.75, P = 0.049; recessive model: OR = 1.82, 95% CI, 1.20–2.74, P = 0.004). Subjects with allele G of hsa-miR-146a/ rs2910164 also showed increased risk of ischemic stroke (OR = 1.33, 95% CI, 1.09–1.62, P = 0.005). Stratification analysis showed that the association between rs2910164 and the risk of ischemic stroke was more pronounced in subjects over 60 years old, females, non-drinkers, subjects without hypertension or diabetes mellitus. There were significant combined effects between miR-146a/rs2910164 and fasting glucose/low-density lipoprotein cholesterol levels on ischemic stroke susceptibility. However, we failed to find any association between the alleles/genotypes of rs11614913 T/C and ischemic stroke, respectively (P> 0.05). In summary, this study provides evidence that miR-146a/rs2910164 might be associated with a significantly increased risk of ischemic stroke in a Chinese population, and the combined effects between miRNA polymorphism and fasting glucose /blood lipid levels may contribute to stroke pathogenesis.

FOXO3a is involved in the apoptosis of naked oocytes and oocytes of primordial follicles from neonatal rat ovaries

Inhibition of the forkhead transcription factor, FOXO3a, can promote the transition from primordial to primary follicle and subsequent follicle development in mammalian ovaries. Stem cell factor (SCF) initiates anti-apoptotic signaling from its membrane receptor, c-kit, to Bcl-2 family members through PI3K/AKT in oocytes of primordial follicles. However, whether FOXO3a mediates the apoptosis of naked oocytes and oocytes of primordial follicles remains unknown. In the present study, oocytes from nests and primordial follicles from neonatal rat ovaries were cultured, and oocyte apoptosis was examined using the TUNEL technique. The pro-apoptotic action of FOXO3a and the potential signal transduction pathways were investigated using RT-PCR, Western blot, and immunocytochemistry. Culturing oocytes in the presence of SCF did not affect the level of total FOXO3a protein, but rapidly elevated the level of phosphorylated FOXO3a (indicating functional suppression). As phosphorylated FOXO3a increased, oocyte apoptosis was inhibited. The specific PI3K/Akt inhibitor, LY 294002, abolished the phosphorylation of FOXO3a and the anti-apoptotic action of SCF. SCF down-regulated the expression of p27KIP1 and pro-apoptotic factors such as Bim, Bad, and Bax, and this activity was reversed by LY 294002. SCF up-regulated the expression of MnSOD, which was also inhibited by LY 294002. However, SCF had no effect on Bcl-2 protein. These results suggest that FOXO3a is involved in oocyte apoptosis in the neonatal rat ovary, and the SCF-PI3K/Akt-FOXO3a signaling pathway mediates oocyte apoptosis and primordial follicle formation.

Enzootic angiostrongyliasis in Shenzhen, China.

To the Editor: Angiostrongylus cantonensis is a zoonotic parasite that causes eosinophilic meningitis in humans after they ingest infective larvae in freshwater and terrestrial snails and slugs, paratenic hosts (such as freshwater fish, shrimps, frogs, and crabs), or contaminated vegetables. With the increase of income and living standards, and the pursuit of exotic and delicate foods, populations around the world have seen angiostrongyliasis become an important foodborne parasitic zoonosis (1–9). Shenzhen municipality is situated in the most southern part of mainland People’s Republic of China between the northern latitudes of 22°27′ to 22°52′ and eastern longitudes of 113°46′ to 114°37′; it shares a border with the Hong Kong Special Administrative Region, China, in the south. The climate is subtropical, with an average annual temperature of 23.7°C. The city is 1,952.84 km2 and has a population of 10 million. Since 2006, thirty-two sporadic cases of human eosinophilic meningitis caused by consumption of undercooked aquacultured snails have been documented in Shenzhen (Shenzhen Center for Disease Control and Prevention, unpub. data). To identify the source of these infections and assess the risk for an outbreak of eosinophilic meningitis, we conducted a survey to investigate whether A. cantonensis occurs in wild rats and snails in Shenzhen. To examine A. cantonensis infection in intermediate host snails, 302 terrestrial snails (Achatina fulica) were collected from 10 investigation sites across Shenzhen, and 314 freshwater snails (Pomacea canaliculata)were sampled from 6 investigation sites. We examined the snails for A. cantonensis larvae by using pepsin digestion standardized procedures (3). To survey the prevalence of adult A. cantonensis in definitive host rats, we collected 187 Rattus norvegicus rats and 121 R. flavipectus rats collected from 4 sites where positive snails positive for A. cantonensis were found. These rats were examined for the presence of adult A. cantonensis in their cardiopulmonary systems. A. cantonensis larvae were found in 96 (15.6%) of 616 examined snails. Of these, P. canaliculata had an average infection rate of 20.7% (65/314), significantly higher (p<0.01) than that of A. fulica (10.3%, 31/302), an indication that P. canaliculata may be the principal intermediate host for A. cantonensis in Shenzhen. A. cantonensis adults were recovered from the cardiopulmonary systems of 37 (12%) of 308 examined rats. Infection rate for R. norvegicus rats was 16.6% (31/187), significantly higher (p<0.01) than that for R. flavipectus (4.9%, 6/121), an indication that R. norvegicus may be the principal definitive host for A. cantonensis in Shenzhen, possibly due to the rat’s preference for eating snails. Infection rates were higher for female rats (25.6% for R. norvegicus and 7.8% for R. flavipectus) than for male rats (8.9% for R. norvegicus, 2.9% for R. flavipectus), possibly because female rats eat more snails to supply proteins for reproduction. This report of enzootic A. cantonensis infection in wild rats and snails in Shenzhen demonstrates the existence of natural origins of infection with A. cantonensis for humans in this city. Persons in Shenzhen eat raw or undercooked freshwater and terrestrial snails and slugs. This practice provides opportunities for infection with A. cantonensis, particularly given that P. canaliculata has been aquacultured intensively for human consumption. The prevalence of A. cantonensis in wild rats and snails in Shenzhen poses substantial risk for future outbreaks of human eosinophilic meningitis. Moreover, public health officials, epidemiologists, researchers, clinical technicians, medical practitioners, parasitologists, and veterinarians, as well as the general public, should be aware of such risks, and integrated strategies should be taken to reduce or eliminate such risks.

Differentially expressed genes between female and male adult Anopheles anthropophagus

The aim of the present study was to identify sex-specific genes in adult Anopheles anthropophagus. As the major malaria vector and Brugia malayi vector in the Asian continent, female Anopheles mosquitoes take blood meals and transmit pathogens through this pathway, while males are nectar feeders. This complex behavior is controlled at several levels, but is probably initiated by the genetic background difference between these two groups. In our study, a subtractive cDNA library for female A. anthropophagus was constructed using the suppression subtractive hybridization (SSH) technique and then 3,074 clones from the female SSH library were analyzed using a microarray-based survey. Genes that were expressed differentially according to sex in A. anthropophagus were screened using real-time polymerase chain reaction and reverse transcription polymerase chain reaction. In our results, we report a series of genes which may be involved in female-specific mosquito behavior, including an inorganic phosphate transporter, a serine protease, the salivary protein GP35-2, and the D7 cluster salivary protein. These findings will provide clues to the nature of insect vectors and open up unprecedented opportunities to develop novel strategies for the control of mosquito-borne diseases.

Monoclonal antibodies against excretory/secretory antigens of Angiostrongylus cantonensis.

In the present study, four murine monoclonal antibodies (MAbs) were generated against the excretory/secretory (ES) products of Angiostrongylus cantonensis adult worms; two represented IgG1 and two represented IgM MAbs, and they were designated 12D5, 15F8, 21B7 and 14G10, respectively. Immunoblotting revealed that all of the MAbs predominantly recognized a 98 kDa antigen in the ES products of A. cantonensis adult worms, and no cross reactions were found with the whole worm antigens of some other common parasites, namely, Schistosoma japonicum, Clonorchis sinensis, Paragonimus westermani, Ascaris lumbricoides, Trichinella spiralis, Anisakis sp., Echinococcus granulosus, Taenia solium, and Spirometra erinacei. Immunolocalization showed that all of the four MAbs reacted with the cuticle of the adult parasite, the external surface of its intestinal canal and reproductive organs, and its egg and first-stage larvae in the lungs of rats experimentally infected with A. cantonensis. The generation and characterization of four specific MAbs against A. cantonensis ES antigens provide foundation for the development of specific immunological diagnostic techniques for human infections with A. cantonensis.

Association between two key SNPs on chromosome 12p13 and ischemic stroke in Chinese Han population.

Objective Genome-wide single nucleotide polymorphism (SNP) association studies recently identified two SNPs (rs11833579 and rs12425791) on chromosome 12p13 that are associated with ischemic stroke (IS) in Caucasian or Black persons from America and the Netherlands. Our aim was to determine whether these SNPs were associated with IS in Chinese Han population. Methods We used a case–control study involving 648 IS patients and 648 age-matched, sex-matched, and ethnicity-matched non-IS controls from two ethnic populations and determined the genotypes of two polymorphisms by TaqMan SNP genotyping assays to assess any links with IS. Results Significant allelic association was identified between rs11833579 and IS in the Han population (odds ratio=1.27, 95% confidence interval=1.08–1.49). One risk haplotype (A-G; odds ratio=1.52, 95% confidence interval=1.21–1.92) was identified in the Han population. Genotypic association analysis demonstrated that rs11833579 confers susceptibility to IS only in a recessive model (P=0.004) rather in additive model. However, the association between rs12425791 and IS was insignificant in Chinese Han population. Conclusion The A allele of SNP rs11833579 on chromosome 12p13 may play a role in mediating susceptibility to IS in the Han Chinese population in a recessive model. The A-G haplotype is also significantly associated with higher IS risk in the Han Chinese population. However, larger populations are warranted to validate our findings.

Protective Effects of Extracts from Fructus rhodomyrti against Oxidative DNA Damage In Vitro and In Vivo

Objective. To evaluate the potential protective effects of extracts from Fructus rhodomyrti (FR) against oxidative DNA damage using a cellular system and the antioxidant ability on potassium bromate- (KBrO3-) mediated oxidative stress in rats. Methods. The effects of FR on DNA damage induced by hydrogen peroxide (H2O2) were evaluated by comet assay in primary spleen lymphocytes cultures. The effects of FR on the activities of SOD, CAT, and GPx and the levels of GSH, hydroperoxides, and 8-OHdG were determined in the plasma and tissues of rats treated with KBrO3. Results. FR was shown to effectively protect against DNA damage induced by H2O2   in vitro, and the maximum protective effect was observed when FR was diluted 20 times. Endogenous antioxidant status, namely, the activities of SOD, CAT, and GPx and the levels of GSH were significantly decreased in the plasma, the liver, and the kidney of the KBrO3-treated rats, while the pretreatment of FR prevented the decreases of these parameters. In addition, the pretreatment of FR was also able to prevent KBrO3-induced increases in the levels of hydroperoxides and 8-OHdG in the plasma, the liver, and the kidney in rats. Conclusions. Our findings suggested that FR might act as a chemopreventive agent with antioxidant properties offering effective protection against oxidative DNA damage in a concentration-dependent manner in vitro and in vivo.

DEHP induces obesity and hypothyroidism through both central and peripheral pathways in C3H/He mice.

Di(2‐ethylhexyl) phthalate (DEHP) is reported to cause obesity and hypothyroidism in both humans and rodents, but the underlying mechanisms were largely unknown. This study was designed to clarify the effects and the mechanisms of DEHP on the pathogenesis of obesity and hypothyroidism and to discover the relationship between them.